Cellular Immune Responses to the Hepatitis B Virus
Polymerase
1,2
Eishiro Mizukoshi,
3* John Sidney,
†Brian Livingston,
‡Marc Ghany,* Jay H. Hoofnagle,*
Alessandro Sette,
†and Barbara Rehermann
4*
CD4ⴙT cells play an important role in hepatitis B virus (HBV) infection by secretion of Th1 cytokines that down-regulate HBV replication, and by promoting CD8ⴙT cell and B cell responses. We have identified and characterized 10 CD4ⴙT cell epitopes within polymerase and used them to analyze the immunological effects of long-term antiviral therapy as compared with sponta-neous recovery from HBV infection. Candidate epitopes were tested for binding to 14 HLA-DR molecules and in IFN-␥ELISPOT and cytotoxicity assays using peripheral blood lymphocytes from 66 HBV-infected patients and 16 uninfected controls. All 10 epitopes bound with high affinity to the most prevalent HLA-DR Ags, were conserved among HBV genomes, and induced IFN-␥ responses from HBV-specific CD4ⴙT cells. Several epitopes contained nested MHC class I motifs and stimulated HBV-specific IFN-␥production and cytotoxicity of CD8ⴙT cells. HBV polymerase-specific responses were more frequent during acute, self-limited hepatitis and after recovery (12 of 18; 67%) than during chronic hepatitis (16 of 48 (33%);pⴝ0.02). Antiviral therapy of chronic patients restored HBV polymerase and core-specific T cell responses during the first year of treatment, but thereafter, responses decreased and, after 3 years, were no more frequent than in untreated patients. Decreased T cell responsiveness during prolonged therapy was associated with increased prevalence of lamivudine-resistant HBV mutants and increased HBV titers. The data provide a rationale for the combination of antiviral and immunostimulatory therapy. These newly described HBV polymerase epitopes could be a valuable component of a therapeutic vaccine for a large and ethnically diverse patient population. The
Journal of Immunology,2004, 173: 5863–5871.
T
he hepatitis B virus (HBV)5is a noncytopathic DNA virusthat causes both acute and chronic liver disease. Despite the availability of an effective vaccine for more than two decades, hepatitis B remains one of the 10 most common causes of death worldwide. More than 5% of the world population, i.e., 400 million people, are currently infected with HBV, and⬎250,000 people die each year from HBV-related liver cirrhosis and hepa-tocellular carcinoma (1). The development of antiviral and immu-nostimulatory therapies for HBV-infected patients therefore re-mains an important priority.
Recovery from acute hepatitis B is the result of a combination of cellular and humoral immune responses. Whereas neutralizing Abs against hepatitis B (HB) virus surface (s) Ag (HBsAg) appear after recovery, cellular immune responses are generally detectable be-fore the synthesis of neutralizing Abs (2) and precede the rise of
serum alanine aminotransferase (ALT) levels (3) as well as clear-ance of HBeAg and HBsAg. They are mediated by cells of the innate immune response (4, 5) and by CD4⫹and CD8⫹T cells that clear HBV by cytolytic (6) and noncytolytic, cytokine-medi-ated (4, 7) mechanisms. After recovery, HBV-specific T cells per-sist in the blood for decades (8). In contrast, in chronic HBV in-fection, HBV-specific cellular immune responses are typically weak, narrowly focused, and rarely detectable in the peripheral blood (2, 9 –11). However, they are not completely absent, because transient increases in the cellular immune response have been shown to precede increases of ALT activity and can be followed by the development of neutralizing Abs and spontaneous recovery (12). Therefore, it has been suggested that therapeutic induction of HBV-specific cellular immune responses may lead to recovery from chronic hepatitis B, and a lipopeptide-based vaccine with an HLA-A2-restricted HBV CD8⫹T cell epitope and a tetanus toxoid CD4⫹T cell epitope has been evaluated as experimental vaccine (13, 14). Although CD8⫹ T cell responses against the HBcore peptide could be induced in healthy, uninfected controls (15) as well as in patients with chronic hepatitis B (16), this was not suf-ficient to mediate viral clearance.
Because HBV-specific CD4⫹ T cells contribute to induction (17) and maintenance of Ag-specific CD8⫹T cells, license den-dritic cells to activate CD8⫹effector T cells (17, 18), and provide help for activation and differentiation of B cells, the induction of HBV-specific CD4⫹T cells is regarded as an important compo-nent of any immunomodulatory therapy. Whereas CD4⫹ T cell epitopes have been identified in the HBcore, HBs, and HBe Ag, the largest protein, HBV polymerase, has not been studied. This is a significant omission because HBV polymerase is a highly immuno-genic CD8⫹T cell target in acute self-limited hepatitis, even though it is produced in significantly smaller quantities than the other HBV Ags (2). Because polymerase is essential for the earliest steps in the
*Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892;†
La Jolla Institute for Allergy and Immunology, and‡
Epim-mune, Inc., San Diego, CA 92121
Received for publication August 19, 2003. Accepted for publication August 20, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was funded in part by National Institutes of Health-National Institute of Allergy and Infectious Diseases Contract N01-AI-95362.
2
This is Publication Number 541 from the La Jolla Institute for Allergy and Immunology.
3
Current address: First Department of Internal Medicine, Kanazawa University School of Medicine, Kanazawa, Ishikawa 920-8641, Japan.
4
Address correspondence and reprint requests to Dr. Barbara Rehermann, Liver Dis-eases Section, National Institute of Diabetes and Digestive and Kidney DisDis-eases, National Institutes of Health, 10 Center Drive, Room 9B16, Bethesda, MD 20892. E-mail address: Rehermann@nih.gov
5
Abbreviations used in this paper: HBV, hepatitis B virus; HCV, hepatitis C virus; HB, hepatitis B; c, core; s, surface; ALT, alanine aminotransferase.
HBV life cycle, recognition of this Ag may limit early HBV spread, and its high degree of conservation may prevent viral escape via mutations in T cell epitopes. Finally, only polymerase-encoding plasmids and not envelope-encoding plasmids were able to break tolerance and to induce specific CD8⫹T cells upon immunization of HBV transgenic mice (19). The characterization of CD4⫹ T cell responses against HBV polymerase, which is performed in the current study, does therefore fill an important gap in our understanding of the HBV-specific immune response and is relevant for the development of multiepitope, immunostimulatory vaccines.
A second issue that is addressed by the current study is the effect of antiviral therapy on T cell responsiveness. Whereas some stud-ies reported a restoration of HBV-specific T cell responses when persistently infected patients were treated with nucleoside analogs that inhibit HBV replication (20, 21), these results have not been confirmed in other studies (22), and most treated patients do not maintain viral control (23, 24). Thus, the hypothesis that high lev-els of HBV may suppress HBV-specific T cell responses is still controversial. Studying 66 patients with past or present HBV in-fection, we show that HBV polymerase-specific responses corre-lated with clinical and serological recovery from hepatitis B and with the duration and outcome of antiviral therapy. Thus, moni-toring HBV polymerase-specific CD4⫹ T cells is of significant value in a diagnostic sense and in immunopathology studies in-vestigating host-virus interaction. In addition, the data provide a rationale for the combination of antiviral and immunostimulatory therapy, and the described HBV polymerase epitopes could be a valuable component of such therapy for a large and ethnically diverse patient population.
Materials and Methods
Patient populationSixty-six adult patients with past or present HBV infection were grouped as follows: group 1, 1 patient with acute, self-limited hepatitis B, who was positive for HBsAg and later seroconverted to anti-HBe and anti-HBs; group 2, 17 recovered patients who were negative for HBsAg, but reactive for anti-HB core (c)⫹anti-HBs⫹; group 3, 14 chronically infected patients who were positive for HBsAg and who were not being treated with lami-vudine; and group 4, 34 chronically infected patients who were positive for HBsAg and who were receiving lamivudine treatment. Groups 3 and 4 were subdivided into patients with (groups 3a and 4a, respectively) or without HBeAg in serum (groups 3b and 4b, respectively). No patient had clinically decompensated cirrhosis. All subjects had been followed in the Liver Diseases Section, NIDDK, National Institutes of Health for ⬎2 years, tested negative for Abs to HIV and hepatitis C virus (HCV), and gave written informed consent to this institutional review board-approved study. Preliminary results of the outcome of lamivudine therapy (100 mg daily) in a subgroup of these patients have been reported (25). Sixteen
healthy blood donors without any history of hepatitis and without HBsAg or anti-HBc in serum served as controls.
Laboratory and virologic testing
Serum HBsAg, HBeAg, and anti-HBe were detected with commercial im-munoassays (Abbott Laboratories, Abbott Park, IL). Molecular typing of HLA DR alleles was performed on genomic DNA using standard site-specific oligonucleotide PCR. Serum HBV DNA was quantitated by branched DNA signal amplification assay (Chiron Corporation, Em-eryville, CA) with a lower limit of detection of 0.7⫻106genome
equiv-alents (106genome equivalents⫽1 mEq) per milliliter (26). Samples with
HBV DNA of⬍1 mEq/ml were tested by quantitative PCR (National Ge-netics Institute, Los Angeles, CA) with a lower limit of detection of 100 copies/ml and by qualitative nested PCR as described (8). RFLP assay was used to detect virological lamivudine resistance defined by either YIDD mutants (methionine-to-isoleucine substitution at codon 552 (M552I)) or YVDD mutants (methionine-to-valine substitution at codon 52 (M552V)) in conjunction with a leucine-to-methionine substitution at codon 528 (L528M) of the HBV polymerase gene (24, 27, 28) as previously described (25). The lower limit of detection of this method was⬃500 copies viral DNA/ml serum.
Synthetic peptides
MHC class II-restricted peptides were synthesized at Epimmune on an Applied Biosystems (Foster City, CA) 430A peptide synthesizer using 9-fluorenylmethoxy carbonyl chemistry and purified to⬎95% purity by reverse-phase HPLC. MHC class I-restricted peptides were synthesized at Research Genetics (Huntsville, AL) at⬎80% purity.
Purification of HLA class II molecules
HLA class II molecules were purified from the EBV-transformed homozygous cell lines LG2 (allele DRB1*0101 (Ag DR1)); MAT (DRB1*0301 (DR3)); PREISS (DRB1*0401 (DR4w4)); KT3 (DRB1*0405 (DR4w15)); SWIG (DRB1*1101 (DR5w11)); Herluf (DRB1*1201 (DR5w12)); HO301 (DRB1*1302 (DR6w19)); PITOUT (DRB1*0701 (DR7)); OLL (DRB1*0802 (DR8w2)); HID (DRB1*0901 (DR9)); GM3107 (DRB5*0101 (DR2w2a)); TR81.9 (DRB3*0101 (DR52a)); L257.6 (DRB4*0101 (DRw53)); and from the transfected fibroblast line L466.1 (DRB1*1501 (DR2w2b)) (29). Large quantities of cells were grown in spinner cultures in RPMI 1640 with 2 mM
L-glutamine (Invitrogen Life Technologies, Carlsbad, CA), 100g/ml
strep-tomycin, 100 U/ml penicillin (Irvine Scientific, Santa Ana, CA), 50M 2-ME, and 10% heat-inactivated FCS (Irvine Scientific), and lysed for 30 min at 4°C with 50 mM Tris-HCl (pH 8.5), 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaCl, and 2 mM PMSF (Calbiochem, La Jolla, CA). Lysates were cleared of debris and nuclei by centrifugation at 15,000⫻g for 30 min. Class II molecules were purified by affinity chromatography using the mAb LB3.1 coupled to Sepharose CL-4B beads as previously described (30, 31).
HLA-DR peptide-binding assays
Fourteen different purified human HLA-DR molecules (5–500 nM) were incubated with various unlabeled HBV peptides and 1–10 nM125
I-radio-labeled probe peptides for 48 h. Assays were performed at pH 7.0 with the exception of that for DRB1*0301, which was performed at pH 4.5.
Table I. MHC class II-restricted peptides
Peptide Number HBV Protein Amino Acid Position Amino Acid Sequencea Conservationb(%)
1 Nuc 50 –70 PHHTALRQAILCWGELMTLA 90 2 Env 180 –195 AGFFLLTRILTIPQS 80 3 Env 339 –354 LVPFVQWFVGLSPTV 95 4 Pol 96 –111 VGPLTVNEKRRLKLI 60 5 Pol 145–160 RHYLHTLWKAGILYK 100 6 Pol 385– 400 ESRLVVDFSQFSRGN 45 7 Pol 412– 427 LQSLTNLLSSNLSWL 90 8 Pol 420 – 435 SSNLSWLSLDVSAAF 85 9 Pol 501–516 LHLYSHPIILGFRKI 80 10 Pol 618 – 633 KQCFRKLPVNRPIDW 45 11 Pol 664 – 679 KQAFTFSPTYKAFLC 60 12 Pol 694 –709 LCQVFADATPTGWGL 95 13 Pol 767–782 AANWILRGTSFVYVP 70 a
Amino acid sequences of nested MHC class I binding motifs are underlined.
b
HLA-DR peptide complexes were separated from free peptide by gel fil-tration on TSK200 columns (TosoHaas, Montgomeryville, PA), and the fraction of bound peptide was calculated as previously described (31). In preliminary experiments, the titers of the HLA-DR preparation were termined in the presence of fixed amounts of radiolabeled peptides to de-termine the concentration of HLA-DR molecules necessary to bind 10 – 20% of the total radioactivity. Peptide inhibitors were typically tested at concentrations ranging from 1.2 ng/ml to 120g/ml. All subsequent in-hibition and direct binding assays were then performed using these HLA-DR concentrations.
The radiolabeled probes were HA Y307-319 for DRB1*0101; TT830-843 for DRB5*0101, DRB1*1101, DRB1*0701, DRB1*0802, and DRB1*0901; MBP Y85-100 for DRB1*1501; MT 65 KD Y3-13 with Y7 replaced with F for DRB1*0301; a nonnatural peptide with the sequence YARFQSQTTLKQKT for DRB1*0401 and DRB1*0405; a nonnatural peptide with the sequence YARFQRQTTLKA for DRB4*0101 (29); a nat-urally processed peptide of unknown origin eluted from a DRB1*1201ⴙ CIR cell line with the sequence EALIHQLKINPYVLS (32), an analog of TT830-843 with the sequence QYIKANAKFIGITE for DRB1*1302 (33); and integrin 3Y24-37 with the sequence YAWASDEALPLGSPR for
DRB3*0101.
ELISPOT assay
IFN-␥ELISPOT assays were performed as previously described with du-plicate cultures of 3⫻105freshly isolated PBMC (34), 10g/ml peptides,
1g/ml HBV core protein (ViroGen, Watertown, MA), 50g/ml tetanus toxoid (University of Massachusetts Medical School, Jamaica Plain, MA), or 1g/ml PHA (Murex Biotech Limited, Dartford, U.K.) in RPMI 1640 containing 5% AB serum and 2 mML-glutamine. Stimulation with PHA always resulted in a vigorous response with spots too numerous to count. In selected experiments, 105CD4⫹or CD8⫹T cells, purified with anti-human CD8 Ab-conjugated magnetic microbeads (Dynal, Oslo, Norway), were cultured with 2⫻105autologous irradiated (3000 rad) PBMCs as
APCs in the presence or absence of Ags. Control experiments confirmed that the irradiated PBMC fraction did not produce any detectable IFN-␥ spots. The number of specific spots (spots in the presence of Ag minus spots in the absence of Ag) was determined with a KS ELISPOT reader (Zeiss, Thornwood, NY), and represented the number of spots in the ab-sence of Ag from the number of spots in the preab-sence of Ag.
PBMC of 16 healthy, anti-HCV negative control persons who were negative for serum HBV DNA, HBsAg, and anti-HBc were tested with the same peptides with the following results (mean number of spot-forming cells⫹SD per 3⫻105PBMC): peptide 1, 1.31⫾2.4; NS3, peptide 2,
0.56⫾1.2; peptide 3, 2.25⫾4.2; peptide 4, 1⫾1.2; peptide 5, 1.25⫾ 2.7, peptide 6, 0.31⫾1; peptide 7, 1.94⫾2.8; peptide 8, 0.44⫾1.1; peptide 9, 2.06⫾2.8; peptide 10, 2.56⫾4.1; peptide 11, 0.31⫾0.8; and peptide 12, 0.81⫾1.4. A response was scored as positive if it was 1) greater than the mean response plus 2 SD in healthy, anti-HCV negative control subjects and 2) greater than 10 specific spots (spots in the presence of Ag minus spots in the absence of Ag) (35).
Cytotoxicity analysis using nested CD8⫹T cell epitopes
HBV-specific CD8⫹T cells were expanded from PBMC by stimulation with the nested peptides that contained HLA-A2 and HLA-A3 binding motifs (Table I). Cytotoxicity was assessed after 3 wk of culture using 25-Ci-labeled, peptide-pulsed HMYC1R cells transfected with HLA-A2.1 or autologous PHA-stimulated T cell blasts as previously described (34). Spontaneous release was ⬍15% of maximum release in all experiments.
Statistical analysis
Fisher’s exact test (two-tailed) was used to compare the frequency of im-mune responses in different patient groups. Student’sttest (two-tailed) was used to compare the level of ALT and HBV DNA in different patient groups. A value ofp⬍0.05 was considered significant.
Results
Selection of highly cross-reactive HLA-DR binding peptides Ten HBV polymerase peptides that contained either the HLA-DR supermotif (36) (peptides 5, 7, 9 –11, and 13) or the DR3 motif (37) (peptides 4, 6, 8, and 12) were tested for binding to a panel of HLA DR B1, B3, B4, and B5 alleles (38). These HLA-DR mol-ecules were selected as representative of the most common HLA variants expressed in the majority of the world’s ethnic population
Table II. HLA-DR binding af finity Peptide Number HLA-DR Binding Capacity (IC 50 nM) a DR Bound b DRB1*0101 DRB1*0301 DRB1*0401 DRB1*0405 DRB1*0701 DRB1*0802 DRB1*0901 DRB1*1101 DRB1*1201 DRB1*1302 DRB1*1501 DRB3*0101 DRB4*0101 DRB5*0101 n % Tested 1 70 — c 85 — 676 196 2273 — 263 193000 9 —— 211 77 8 2 1 — 8 253 8 188 58 — 6 1 0 217 — — 1053 9 9 0 3 385 — 300 27 2717 74 30 — 53 1944 13 — — 1429 7 7 0 4 8415 43 3916 1908 — 5354 — 6666 — 4461 4153 — 8121 4330 1 1 0 5 1 7 — 2271 1499 61 36 133 42 149 766 4 — 782 35 10 83 6 7372 36 208 251 ————— 946 1368 2525 8711 — 4 50 7 2 — 10 47 173 598 791 303 397 143 21 1837 4179 1067 10 77 8 38 62 168 17 5063 1065 7126 4923 1859 36 3089 57 —7 5 4 9 248 — 77 244 — 800 1551 492 9462 — 558 — 102 560 88 0 10 3 — 40 34 62 872 5175 1617 — 821 4370 — 3060 1246 6 5 5 11 10 — 88 181 90 416 142 82 — 190 41 4848 322 144 11 92 12 7470 67 490 1203 ————— 2022 5009 1808 1044 — 2 25 13 55 — 966 1634 44 214 299 1520 802 143 386 — 6553 3279 8 6 7 aIC 50 represents the amount of peptide required for 50% inhibition of binding of the fluorescein-labeled reference peptide to each HLA-DR molecule. bNumber of DR alleles bound ⱕ 1000 nM. The binding capacities ⬍ 1000 nM are indicated in bold. c—, Indicates an IC 50 of ⬎ 20 M.
(38). The sequences of most selected HBV polymerase peptides were conserved in ⬎80% of the 20 most common HBV strains including adr, adw, ayr, and ayw isolates (Table I). In addition, several peptides that were conserved in only⬃50% of isolates, but highly conserved in their DR binding region, were included in the analysis. For comparison, we also studied one HBV nucleocapsid (39) and two HBV envelope peptides (40, 41) (Table I) and the HBcore protein (see Fig. 4).
As shown in Table II, all HBV peptides bound with an IC50of ⱕ1000 nM to at least one HLA-DR molecule. The IC50 value represents the amount of peptide required for 50% inhibition of binding of a fluorescein-labeled reference peptide. An IC50value of 1000 nM has previously been shown to represent the affinity threshold associated with immunogenicity (38, 42). Moreover, 8 of the 10 polymerase peptides bound at least four of these frequent DR molecules. This degree of cross-reactivity is not dissimilar from the previously identified control peptides (39 – 41), and sug-gested that these peptides should be recognized by subjects with diverse HLA haplotypes. Based on the worldwide prevalence of the HLA-DR Ags, this panel of peptides should cover 98.2% of the average population.
Immunogenicity of HBV polymerase peptides as assessed by IFN-␥ELISPOT analysis
To determine whether these highly cross-reactive and conserved HLA-DR binding peptides were recognized by T cells of patients with past or present HBV infection, IFN-␥ELISPOT responses were evaluated with PBMC from 1 acutely HBV-infected patient, 17 recovered patients, and 48 persistently HBV-infected patients (Table III). Sixteen blood donors were tested as controls. The cut-off of positivity was set atⱖ10 IFN-␥spots/300,000 PBMC (see Materials and Methods). All 10 HBV polymerase peptides were recognized by PBMC of at least one patient, and 29 of 66 patients (44%) responded to at least one of the analyzed HBV polymerase peptides (Fig. 1). Peptides 4 and 8 were the only peptides that were exclusively recognized by the patient with strongest immune re-sponse and acute hepatitis B, and one of these peptides (peptide 4) had a very low HLA-DR binding affinity (Table II). By contrast, peptides 7 and 11, associated with significant binding to 77 and 92% of the HLA-DR molecules, respectively, were recognized by 8 and 13 patients. These data demonstrate that conserved HBV polymerase peptides selected on the basis of a high binding affinity to several HLA DR molecules are antigenic in the context of mul-tiple DR alleles, and thus allow coverage of a broad and ethnically diverse patient population.
In the total patient population, 61 responses were observed against HBV polymerase peptides. The patients’ HLA haplotypes
were known for 56 of these 61 cases. When the observed responses were evaluated in the context of the patients’ HLA haplotypes, 55% (31 of 56) of all responses occurred in the presence of one of the DR alleles with high peptide-binding affinity (IC50⬍1000 nM; Table II). In an additional 20% (11 of 56) of all responses, the patients expressed DR molecules at least weakly binding (1000 nM⬍IC50⬍20M) the peptide. In the remaining cases, patients did not express any DR alleles known to bind the peptide (IC50⬍ 20M; Table II), suggesting that some of the responses might be restricted by other alleles not analyzed in the current study.
The magnitude of the HBV-specific T cell responses was as-sessed by the frequency of HBV nucleocapsid, envelope and poly-merase peptide-specific, IFN-␥-producing T cells in the PBMC population (Fig. 2). The broadest T cell response was noted during acute, self-limited hepatitis B with responses to all 13 HBV epitopes. In contrast, none of the other patients responded to more than five epitopes, and the overall response of patients with chronic hepatitis B was weak. As previously reported for other HBV epitopes and proteins (35, 43, 44), the frequency of HBV epitope-specific cells was low, i.e., between⬃10 and 50 peptide-specific cells per 300,000 PBMC. Tetanus toxoid-specific responses were tested as a positive control, and the frequency of responses did not differ significantly among patient subgroups (Fig. 2C).
T cell subsets responding to HBV polymerase peptides
To determine the T cell subset that responded to the HBV poly-merase peptides, PBMC were separated into CD4ⴙ and CD8ⴙ subpopulations using Ab-coated magnetic beads. Each cell sub-population was then separately tested in the presence of irradiated, autologous APCs and the respective peptides. Fig. 3A demon-strates in a representative experiment that most IFN-␥spots in the ELISPOT assay segregated with the CD4ⴙ T cell subfraction. However, in the case of peptide 9,⬃25% of the total number of IFN-␥spots could still be attributed to CD8ⴙT cells (Fig. 3A). A closer analysis of the peptide sequences revealed that peptide 9 as well as four other polymerase peptides contained nested HLA class I-binding motifs (Table I). In fact, nested sequences within pep-tides 5, 6, and 11 have previously been described as CD8ⴙT cell epitopes (45). Thus, in natural HBV infection, these shorter pep-tides may be endogenously processed from polypeptide precursors, loaded onto HLA class I molecules in HBV-infected cells, trans-ported to the cell surface, and stimulate responses of CD8ⴙT cells. To test this hypothesis, we chose the nested sequences HLY-SHPIIL in peptide 9 and QAFTFSPTYK in peptide 11, which contained the HLA-A2 and HLA-A3 binding motifs, respectively, and tested them for CTL recognition using a microwell peptide stimulation technique to expand low-frequency HBV-specific CTL
Table III. Baseline characteristics of the patients studied
Group Clinical Diagnosis
No. of Patients
Sex
M/Fa Race C/A/
AA/Mb Age (yr)
Mean⫾SD ALT (IU/L) Mean⫾SD HBsAg⫹ n(%) HBV DNA (copies/ml) 1 Acute HBV patient 1 1/0 1/0/0/0 27 641 1 (100) 1.5⫻104 2 Recovered patients 17 15/2 14/1/2/0 52⫾9 55⫾12 0 (0) 0c
3 Chronic hepatitis B, no therapy
3a HBeAg positive 7 6/1 4/2/0/1 45⫾21 97⫾58 7 (100) 3.2⫻108⫾2.8⫻108
3b HBeAg negative 7 4/3 3/4/0/0 49⫾9 87⫾87 7 (100) 2.5⫻107⫾5.2⫻107
4 Chronic hepatitis B, on therapyd
4a HBeAg positive 20 18/2 13/6/1/0 46⫾3 84⫾82 20 (100) 2.0⫻108⫾2.7⫻108
4b HBeAg negative 14 12/2 5/6/1/2 49⫾13 41⫾21 14 (100) 3.4⫻105⫾9.5⫻105
a
M, Male; F, female.
b
C, Caucasian; A, Asian; AA, African-American; M, Mediterranean.
c
Negative by branched DNA analysis and nested PCR.
d
precursors from PBMC. Overall, the HLA-A2 restricted epitope in peptide 9 and the HLA-A3-restricted epitope in peptide 11 ex-panded HBV-specific CD8ⴙT cells of several patients with the corresponding HLA class I haplotype (Fig. 3B). These results dem-onstrate that several of the newly identified CD4ⴙT cell epitopes contained shorter sequences that were recognized by CD8ⴙT cells when presented as the minimal optimal epitope in the context of the matching HLA class I molecule.
Cross-sectional analysis of HBV polymerase-specific T cell responses in patient groups with different outcome of HBV infection
Because HBV polymerase is an essential enzyme for HBV repli-cation, expressed early in the HBV life cycle and also found inside the secreted HBV particle, we reasoned that the frequency of HBV-specific T cell responses might correlate with different stages of HBV infection. As previously shown for CD8ⴙT cell responses against polymerase (2, 3), there was a significant difference in the frequency of CD4⫹T cell responses between the different patient subgroups. Whereas the patient with acute, self-limited hepatitis B recognized all epitopes, and 11 of 17 (65%) recovered patients (group 2) tested positive for at least one HBV polymerase peptide, only 16 of 48 (33%) of patients with chronic hepatitis B (groups 3 and 4) tested positive (p ⫽ 0.043; Fig. 1). When immune re-sponses against any peptide, i.e., including the nucleocapsid and envelope peptides, were considered, this difference between
acutely infected and recovered patients (16 of 18 (89%) respond-ing) and chronically infected patients (19 of 48 (40%) respondrespond-ing) was even more significant (p⬍0.0001; Fig. 1).
Among patients with chronic hepatitis B, further analysis was performed to assess the role of lamivudine therapy on cellular immune responses to HBV polymerase peptides. Cellular immune responses to HBV polymerase were detected in 4 of 14 untreated (29%) and in 12 of 34 (35%) treated patients. Within the lamivu-dine-treated group, the duration of treatment at the time of testing appeared to correlate with reactivity. Four of 5 (80%) patients treated for⬍1 year responded to the polymerase epitopes as com-pared with 3 of 7 (43%) patients treated for 1–3 years, 5 of 22 (24%) patients treated for ⬎3 years, and 4 of 14 (29%) patients who were not treated (Fig. 4A). These differences in the poly-merase-specific responses between the subgroups were statistically significant (p⫽ 0.029 for⬍1-year treatment vs ⬎3-year treat-ment; p ⫽ 0.041 for ⬍1-year treatment vs ⬎1-year treatment) (Fig. 4A) and correlated with differences in viral levels among patient subgroups. For example, the highest frequency of HBV polymerase-specific T cell responses was observed in patients who had received lamivudine therapy for ⬍1 year and displayed the lowest HBV DNA levels. In contrast, HBV polymerase-specific T cell responses were lower in patients who had been treated for 1–3 and for 3–5 years, respectively, and these patient subgroups were characterized by 100- to 1000-fold higher HBV DNA levels due to development of lamivudine-resistant HBV mutants (Fig. 4A).
FIGURE 1. HBV nucleocapsid, envelope, and polymerase peptide-specific T cell responses. Only patients who recognized at least one of the 13 tested
peptides in the IFN-␥ELISPOT assay are shown. A positive IFN-␥response was defined as⬎10 spot-forming cells per 300,000 PBMC, which is greater than the mean plus 2 SD of the baseline response detected in 16 healthy uninfected controls. Shaded boxes indicate the presence of a significant IFN-␥T cell response to HBV nucleocapsid and envelope peptides; black boxes indicate the presence of a significant IFN-␥T cell response to HBV polymerase peptides. Peptide sequences are described in Table I, and patient groups in Table III.
Overall, the frequency of HBV polymerase-specific responses was higher in patients who had successfully cleared HBeAg (Fig. 4B) than in patients who remained HBeAg positive under lamivu-dine therapy (B). When HBeAg⫹and HBeAg⫺patients were an-alyzed separately, HBV polymerase-specific T cell response de-creased in both subgroups under prolonged lamivudine therapy. Consistent with the findings for all treated patients (group 4), this reduced T cell responsiveness correlated with increased HBV DNA levels and emergence of lamivudine resistance mutants, but did not reach statistical significance due to the small number of patients in each subgroup (Fig. 4B). Finally, a similar trend toward decreased HBV-specific immune responsiveness under prolonged lamivudine therapy was observed for HBcore-specific responses (Fig. 4C), whereas responses to the control Ag tetanus toxoid were comparable for all patients (Fig. 4D).
Collectively, the data suggest that antiviral therapy restores HBV polymerase-specific immune responses transiently, i.e., dur-ing the first year of therapy, and that cellular immune responses gradually attenuate with emergence of lamivudine-resistant HBV mutants and increase of viral levels under more prolonged therapy.
Discussion
The current study uses a combined immunochemical and cellular immunology approach to identify and characterize 10 highly con-served and immunogenic CD4⫹T cell epitopes in the HBV poly-merase protein. The newly identified CD4⫹T cell epitopes within HBV polymerase are relevant for several reasons. First, all epitopes displayed significant binding affinity to multiple HLA-DR molecules. Therefore, the data suggest that the majority of the world’s population, independent of ethnicity, should be able to respond to these epitopes. Second, CD4⫹T cell epitopes within HBV polymerase offer significant advantages for the development of epitope-based diagnostics and vaccines, because of the high
degree of sequence conservation of HBV polymerase and its es-sential role in the viral life cycle, which reduces the possibility of viral escape by mutations in T cell epitopes. In addition, HBV polymerase represents the largest HBV protein and has already been shown to be highly immunogenic at the CD8⫹T cell level in patients with acute, self-limited hepatitis B (2, 3). Third, the newly
FIGURE 2. Vigor of HBV nucleocapsid,
enve-lope, and polymerase peptide-specific T cell re-sponses. Aand B, Direct ex vivo IFN-␥ELISPOT analysis of peripheral blood T cell responses to HBV nucleocapsid and envelope peptides (A) and HBV polymerase peptides (B). Only significant IFN-␥ re-sponses greater than the mean plus 2 SD (indicated by horizontal line) of the baseline response detected in 16 healthy uninfected controls and ⬎10 specific spots/300,000 PBMC are shown. The asterisk indi-cates 102 spots. Peptide sequences are described in Table I, patient groups (u, group 1;f, group 2;䡺, group 3;o, group 4) are defined in Table III.C, Tet-anus-specific responses. Responses of ⬎100 spots were too numerous to count with the automated ELISPOT reader.
FIGURE 3. Identification of nested MHC class I-restricted epitopes.A,
PBMC were sorted into CD4ⴙ and CD8ⴙ T cell subpopulations using Ab-coated magnetic beads and stimulated separately with peptide-pulsed autologous APCs. The number of peptide-specific, IFN-␥-producing cells as determined by ELISPOT analysis is indicated and greater for the CD4ⴙ T cell subpopulation than for the CD8ⴙT cell subpopulation. A represen-tative experiment is shown.B, Cytotoxic CD8ⴙT cells specific for MHC class I-restricted peptides that are nested within the HBV-polymerase CD4ⴙ T cell epitopes. T cell lines were generated from PBMC of the HLA-A2-positive patients 12, 29, and 39, and the HLA-A3-positive patient 17, respectively, by stimulation with the nested 9- and 10-mer peptides located within peptide 9 (F) or 11 (f) (see Table I). Expanded T cell lines were then tested for specific cytotoxicity against the nested peptides in a standard51Cr release assay at the indicated E:T ratios.
identified HBV polymerase epitopes were able to induce IFN-␥ production by HBV-specific T cells, an effector function associ-ated with in vivo suppression of HBV replication (7, 46). Fourth, the observation that several of these CD4⫹T cell epitopes contain nested MHC class I-restricted epitopes that stimulate IFN-␥ re-sponses and cytotoxicity by CD8⫹T cells is an additional attrac-tive feature for a multiepitope vaccine. The well-characterized, immunodominant HBc18 –27CD8⫹ T cell epitope, for example, does also overlap with an MHC class II-restricted CD4⫹T cell epitope (47), and it has been described that CD4⫹ T cells are indispensable for the maintenance of functional CD8⫹T cells that control chronic viral infections (48). This CD4⫹T cell help may consist of direct help for CD8⫹T cells via production of cytokines (49) or of indirect help for professional APCs via CD40/CD40L-mediated activation (50). Moreover, recent studies have demon-strated the need to include potent CD4⫹T cell epitopes to restore an altered Th response and to overcome CD8⫹T cell tolerance in chronic HBV infection (14). Because covalent linkage of CD4⫹ and CD8⫹T cell epitopes on the same peptide vaccine construct is important for the induction of Ag-specific responses (51), the nat-ural occurrence of nested CD8⫹and CD4⫹T cell epitopes within the polymerase Ag might provide an elegant and promising ap-proach to fulfill these requirements.
Finally, the presence of HBV polymerase-specific, CD4⫹T cell responses correlated with different outcomes of HBV infection. As demonstrated for CD8⫹ T cell responses (2), circulating HBV polymerase-specific CD4⫹T cell responses were more frequent in acute self-limited hepatitis B and after recovery than in chronic hepatitis B. Moreover, even in the persistently infected, HBsAg⫹ patient subgroup, HBV polymerase-specific T cell responses were associated with a partial response, i.e., with loss of HBeAg (Fig.
4B) and reduction of HBV DNA levels (A). Although the recog-nition of endogenously processed HBV polymerase could not be tested due to a lack of recombinant and purified polymerase pro-tein, these observations and the absence of those responses in healthy, uninfected blood donors indicated that all epitopes were processed in vivo and that HBV polymerase-specific CD4⫹T cells were primed in vivo.
In the second part of this study, the newly identified CD4⫹T cell epitopes were then used to analyze the HBV-specific immune response during antiviral therapy. Recent studies have suggested that suppression of HBV replication by lamivudine rapidly restores HBV-specific cellular immune responsiveness to the same level as in recovered persons (20). In that study, both CD4⫹ (20) and CD8⫹(21) T cells specific for HBV remained detectable in the blood for up to 5 mo during lamivudine therapy. However, the increased T cell reactivity was not associated with an increase in loss of HBeAg or HBsAg (20), the serological hallmark of recov-ery from hepatitis B. Also, a second study of patients receiving IFN-␣alone and in combination with lamivudine reported no res-toration or de novo induction of HBV core-specific T cell prolif-eration (22). Because HBV polymerase is expressed early in the HBV life cycle and also found inside the secreted HBV particle, we reasoned that the frequency of HBV polymerase-specific T cells might correlate more closely with viral levels and the out-come of lamivudine therapy.
Using the newly identified HBV polymerase peptides, the cur-rent study indicated that antiviral therapy of persistently infected patients appeared to increase the frequency of HBV-specific CD4⫹ T cell responses during the first year of treatment. This result in-dicates that HBV-specific T cells are not completely depleted or anergized during chronic hepatitis B because their frequency in the
FIGURE 4. Prevalence of HBV
polymerase (AandB), HBcore (C), and tetanus toxoid-specific (D) T cell re-sponses in recovered patients (group 2), untreated patients with chronic hepatitis B (group 3), and patients with chronic hepatitis B during lamivudine therapy (group 4). Patients with chronic hepati-tis are separated into those with HBeAg or without HBeAg inB.
peripheral blood can significantly increase upon therapeutic reduc-tion of HBV levels. The reconstitureduc-tion of HBV-specific T cells in the periphery may result from generation of new precursors in the regional lymph nodes on the one hand (52) and from decreased sequestration of effector T cells to the liver.
With longer periods of antiviral therapy, however, the frequency of HBV-specific CD4⫹T cell responses decreased and, after 3 years of therapy, responses were no more frequent than in un-treated patients. Decreased responsiveness of HBV-specific T cells correlated with the emergence of lamivudine-resistant HBV mu-tants and a concomitant increase of HBV DNA levels. Whether the lack of HBV-specific responses predated the development of viral resistance and may have contributed to its development, is an in-teresting question, that could not be answered due to the cross-sectional nature of this study and warrants future, prospective stud-ies. Overall, the results suggest that therapeutic vaccination designed to boost HBV-specific cellular immunity might be most efficient during the first year of antiviral therapy. The degenerate HLA-DR binding and immunogenicity of the HBV polymerase epitopes as well as the inclusion of nested CD8⫹T cell epitopes make these epitopes valuable components of a vaccine that is de-signed to cover a large and ethnically diverse patient population.
Acknowledgments
We thank Ms. Yoon Park for invaluable help with sample collection, and all of the patients who donated blood samples for this study.
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